The methods of recombinant DNA technology have enabled the production of relatively large quantities of biologically important polypeptides and proteins. For much of this work, the bacterium Escherichia coli has been employed as a host organism for the expression of recombinant vectors, but the use of this bacterium has significant limitations. Because of their physical structure, E. coli bacteria expressing genes for recombinant polypeptides or proteins must generally be disrupted by physical, chemical or enzymatic means before the recombinant products can be isolated.
E. coli and other gram negative bacteria are characterized by a central cytoplasm, where proteins are synthesized, and a complex cell membrane structure. There is an external membrane, to which a number of lipid-linked oligosaccharides are bound. When pathogenic gram negative bacteria infect an animal, the production of antibodies specific for these surface oligosaccharides can be important in determining the course of the disease.
Interior to the external membrane is the plasma membrane, which is the major permeability barrier of the cell. This membrane contains proteins that allow certain nutrients and other chemicals to pass into and out of the cell, while excluding others.
Between the external membrane and the plasma membrane is the periplasmic space, or periplasm. This region contacts the outer membrane and contains a peptidoglycan, a highly cross-linked wall-like complex of proteins and oligosaccharides that gives rigidity to the cell.
Most proteins synthesized by E. coli remain in the cytoplasm, but some are found in the periplasm. Proteins which are transported from the cytoplasm to the periplasm contain "signal peptides" which are covalently linked by a peptide bond to the amino termini of the proteins and which facilitate transport through the plasma membrane. Examples of some periplasmic proteins in E. coli are .beta.-lactamase, alkaline phosphatase and certain nucleases, peptidases and proteases. The signal peptides of the periplasmic proteins are generally cleaved at some point during the transport process, leaving the "mature" forms of the proteins in the periplasm.
During the production of recombinant proteins using E. coli, the expression products of heterologous or foreign genes generally accumulate in the cytosol. Such proteins often precipitate to form insoluble "inclusion" or "retractile bodies". Recombinant proteins in such bodies are not in their native conformation and are not biologically active [Mitraki et al., Bio/Technology 7:690 (1989)]. To isolate such proteins in a useful form, the bacteria must be disrupted and the proteins in the insoluble fraction must be solubilized using a detergent or a chaotropic agent such as urea or guanidine-hydrochloride. Because proteins thus solubilized are not in their native conformations, they must be correctly refolded using relatively complex procedures such as those described by Builder et al. (European Patent Application Publication No. 114 506).
In an effort to use recombinant DNA methods to produce heterologous proteins that do not accumulate in cytoplasmic inclusion bodies, Villa-Komaroff et al. [Proc. Natl. Acad. Sci. USA 75:3727 (1978)] inserted the rat preproinsulin gene into the E. coli .beta.-lactamase gene. As already noted, b-lactamase is a periplasmic enzyme which, in its precursor form, carries a signal peptide. The fusion protein resulting from the expression of the fused .beta.-lactamase/preproinsulin genes was transported to the periplasm by the above-described transport mechanism.
Similarly, Gilbert et al., (European Patent Application Publication No. 006 694) have disclosed the production of genetically engineered fusion proteins by expression of DNA sequences containing a gene encoding a desired foreign protein fused to a DNA sequence encoding a signal peptide of a periplasmic protein.
Exploiting the natural transport processes a bit further, Silhavy et al. (U.S. Pat. No. 4,336,336) have described a method for producing fusion proteins which are transported into the outer membrane of a bacterium. This method entails the fusion of a gene encoding a cytoplasmic bacterial protein with a gene for a non-cytoplasmic carrier protein, thereby producing a fusion protein which is carried to the outer membrane. Silhavy et al. also disclose that this method could be used to insert a foreign gene (e.g., a gene encoding a eukaryotic protein) into the already constructed fusion gene.
In none of the foregoing processes, however, are the desired recombinant proteins transported beyond the outer membranes of the cells. In each case, the cells must still be disrupted to recover the proteins. As a result, myriad bacterial proteins are also released, rendering the isolation of the desired proteins more laborious and complex. Moreover, where the processes yield products fused to bacterial proteins, the products must generally be cleaved to produce the desired protein. This process may be complex and may entail the use of denaturing conditions, making recovery of proteins having full biological activity difficult.
More recently, Sakaguchi et al. [Agric. Biol. Chem. 52:2669 (1988)] have reported fusing a DNA sequence encoding the ompA signal sequence to a gene encoding granulocyte-macrophage colony stimulating factor (GM-CSF) in an E. coli expression vector. After transformation into E. coli HB101 and expression, it was found that some GM-CSF was secreted into the culture medium.
E. coli mutants which leak various periplasmic enzymes into the culture medium have been produced. For example, Lopes et al. [J. Bacteriol. 109:520 (1972)] treated E. coli cells with a mutagen such as nitrosoguanidine to produce "periplasmic leaky" mutants which secreted ribonuclease I, endonuclease I and alkaline phosphatase. Similarly, Anderson et al. [J. Bacteriol. 140:351 (1979)] and Lazzaroni et. al. [J. Bacteriol. 145:1351 (1981)] have used immunoprecipitation or SDS-polyacrylamide gel electrophoresis to detect secreted periplasmic proteins in studies of periplasmic leaky mutants.
The leakiness of such mutants is believed to reflect a deficiency in a normal component(s) of the bacterial outer membrane which increases permeability. None of the leaky mutants were made with the objective of obtaining secretion of recombinant proteins into the culture medium. Instead, their construction appears to have been carried out to investigate the structure and function of the bacterial envelope and the location of various enzymes within the membrane structure.
More recently, Zinder al. (U.S. Pat. No. 4,595,658) have disclosed a method for facilitating the externalization of proteins synthesized in bacteria. This method entails the introduction of all or a portion of gene III of an fl bacteriophage into a plasmid or bacterial chromosome. The fl bacteriophage gene III protein produced by expression of the gene is said to perturb the outer bacterial membrane, resulting in the leakage of periplasmic proteins from the cell.
Zinder et al. further disclose that their leaky mutants can be used to produce genetically engineered fusion proteins by a method in which a gene encoding a desired protein is fused to a DNA sequence encoding a leader capable of transporting the protein to the periplasmic space. Zinder et al., however, provide no teachings of how such fusions could be carried out and no example to show that the method would actually work as hypothesized. All that is actually shown is that the natural .beta.-lactamase of the mutants leaked from the cells into the surrounding medium.
Because improper chain folding and protein denaturation are associated with recombinant proteins maturing within the cytoplasm and do not generally occur with proteins exported out of the cell, there is a need for a reliable way to make and use E. coli secretory strains.